This R21 proposal focuses on developing in vivo and in vitro Plasmodium cynomolgi model systems to advance cell biological and biochemical research on P. vivax, a major widespread human malaria pathogen. In particular, the goal is to enable the routine application of transfection technologies that will address hypothesis-driven questions regarding the unique biology held in common between P. vivax and P. cynomolgi, which is not found in the other major human malaria, P. falciparum or any of the popular rodent malaria models. This biology includes, among many other differences, the development and reactivation of dormant liver-stage forms known as hypnozoites, and the synthesis and functioning of numerous caveolae vesicle complexes (CVCs) and other membrane structures in infected red blood cells (iRBCs). While P. vivax cannot be cultured continuously in vitro, to facilitate such experimentation, P. cynomolgi blood-stage parasites can be easily manipulated genetically in vivo and ex vivo from rhesus monkey infections where large quantities of parasites can be obtained as well as be adequately propagated and experimentally manipulated by in vitro culture for several days to weeks. The phylogenetic close kinship and nearly identical basic morphology and biology shared by these two species strongly support the proposed utility of P. cynomolgi model systems. No rodent malaria model or P. falciparum culture system offers any biology that mimics the unique biology of these species, and thus they cannot serve as model systems to forward this research. In Aim 1 we will focus on attaining transformed P. cynomolgi parasites that constitutively throughout the life-cycle or at specific developmental time points express single or dual-color fluorescent tags, as the first step for future studies aiming to identify, purify and investigate specific life cycle stages, with a primary interest at this time on the development and activation of the elusive hypnozoite stage. In Aim 2, transformed parasites will be developed to study the trafficking machinery, pathways and unique membrane structures produced by P. vivax and P. cynomolgi blood-stage parasites, which include the CVCs, extensive networks of cleft membranes and novel proteins catalogued by proteomics, immunochemical studies and microscopy. One specific question to be addressed is how and when the PHIST81-95 protein traffics from the parasite beyond the parasitophorous vacuole membrane to its RBC cytosolic location on the cytoplasmic face of the CVCs. For this, the Conditional Aggregation Domain (CAD) protein regulation system will be applied. Critically, P. cynomolgi parasites provide a superior model for the proposed studies, and development of this model will enable research on initial hypothesis-driven questions posed here and ongoing research to understand and define biological and biochemical targets of intervention for P. vivax.